Thieno- and pyrrolopyrimidine analogues and prodrugs thereof as anticancer agents and methods of use thereof

ABSTRACT

The present invention provides for the design and synthesis of halogenated thieno- and pyrrolopyrimidine compounds and prodrugs thereof that exhibit cancer proliferation inhibitory activity and the use thereof for cancer treatment.

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention is a Continuation-In-Part application claimingpriority to copending U.S. application Ser. No. 15/893,779, filed onFeb. 12, 2018, which is turn is claims priority to U.S. patentapplication Ser. No. 15/249,980, filed on Aug. 29, 2016, now U.S. Pat.No. 9,895,374, which in turn is a continuation of and claims priority toU.S. patent application Ser. No. 14/634,940, filed on Mar. 2, 2015, nowU.S. Pat. No. 9,434,742, the contents of which are incorporated byreference herein for all purposes.

FIELD OF INVENTION

The present invention provides for the design and synthesis ofhalogenated thieno- and pyrrolopyrimidine compounds and prodrugs thereofthat exhibit cancer proliferation inhibitory activity and the usethereof for cancer treatment.

BACKGROUND OF THE INVENTION

The advent of molecularly targeted anticancer therapies has energizedthe cancer field and given new hope to cancer patients. Discovery ofintracellular pathways that can lead to tumor growth suppression and thedevelopment of monoclonal antibodies that specifically target cancerpathways are being developed and tested in human clinical trials. Thedawn of such targeted anticancer therapies could cause a certain lack ofinterest in the development of novel conventional cytotoxic smallmolecule drugs. However, molecularly targeted therapies have not shownimproved curative properties when administered as monotherapy [1]. As aresult, most molecularly targeted agents today are used in combinationwith cytotoxic compounds.

In contrast to the tumor-static activities of many targeted agents,cytotoxic agents have the ability to directly kill cancer cells throughtheir inhibition of critical cell growth pathways and exert a broaderrange of activity as compared to targeted therapies that are inherentlynarrow. For these reasons, there is hope in combining molecular targetedanticancer therapies with conventional cytotoxic chemotherapy [2, 3].Indeed, current standard of care treatments are based on sequentialtherapies by combining multiple drugs with the intent of overcomingdrug-resistance, which is typical of many tumors and a major issue infighting cancer. In the age of targeted therapies and therapeuticmonoclonal antibodies, it is noteworthy that extensive resources andscores of clinical trials are still being devoted to the identificationand evaluation of small molecules chemotherapy agents, thus reiteratingthe need for improved chemotherapy drugs. This is partly due to anextremely large untapped reservoir of potential therapeutic compoundswhich tackle several biological aspects of a highly proliferating livingcell and also to the growing understanding of the life cycle of cancercells and onset of drug-resistance. Overall, there is still a need todevelop more effective cytotoxic drugs. These new chemotherapy drugsneed to show an increased potency, low systemic toxicity, betterbioavailability and that can be used in combination with currentapproved drugs.

Among cancers, the high incidence and mortality of colorectal cancer isa major health issues worldwide [4, 5]. While the ability to treatpatients with colorectal cancer has improved in the past 15 years,thanks to optimized surgery techniques, better radiotherapy and novelchemotherapy drugs, treatments are still not ideal. Major toxicities(grade 3 or 4) are still a problem with standard-of-care chemotherapydrugs. SFU, irinotecan, leucovorin, oxaliplatin, either alone or in theFOLFOX or FOLFIRI regimens, result in severe side effects that can alsodeveloped into neuronal toxicity [6, 7]. Targeted therapies displayedoverall less side effects events when administered alone, however,combinations with chemotherapy drugs is typically more effective incolorectal patients, especially when RAS/RAF mutations are present. Forthe latter, the identification of KRAS and BRAF mutations in colorectalcancer patients has become a crucial diagnostic factor for designingeffective therapy regimens that could treat also metastatic colorectalcancers [8, 9].

Additionally, melanoma cancer rates have risen steadily for the last 30years. Nonetheless, the median survival is <1 year and the 5 yearsurvival rate is 10%. [30, 31]. Two targeted therapies were approved in2011, ipilimumab (a monoclonal antibody) and vemurafenib (a BRAF kinaseinhibitor), that exhibit robust anti-tumor activity, but patientsrelapse with lethal drug resistant disease within 5-7 months.[32-35] Thebiological response modifier adesleukin (recombinant interleukin-2) hasa 50-60% response rate, but can produce life-threatening toxicities. Theresponse rate for temozolomide, a DNA modifying agent, is <20%.

Breast cancer is a major cause of cancer-related death in women precededonly by lung cancer.[43] The expression levels of estrogen receptors(ER) and progesterone receptors (PR) as well as the amplification statusof the HER-2/Neu gene help direct diagnosis and treatment of breastcancer. For tumors that express one or more of these biomarkers,targeted therapies have significantly improved patient outcomes.However, breast cancers lacking the aforementioned biomarkers, termedtriple negative breast cancer (TNBC), present severe challenges forpatient survival since biomarker-targeted therapies are ineffective.

As discussed above, current therapies are not always effective, and assuch, there is a need for improved drugs with higher activity fortreating cancers with minimum side effects that can be used in alone,combinational and/or sequential treatments.

SUMMARY OF THE INVENTION

The present invention provides for nucleobase analogues, that being,halogenated thieno- and pyrrolopyrimidine compounds and prodrugs thereofwith the ability to induce apoptosis and inhibit growth of cancer cellsat low nM concentrations. The activity of these compounds is effectiveagainst numerous types of cancers including colon, renal, breast andmelanoma.

Further, methods of designing prodrugs and of decreasing toxicity animproving safety index by modifying a therapeutic agent to create aprodrug are disclosed. Such modification provides an improvedtherapeutic index as compared to the free therapeutic agent.

The present invention further includes methods of treating a medicalcondition by administering the prodrug of the invention.

The present invention also relates to the induction of cancer cellapoptosis with halogenated pyrrolopyrimidines, thienopyrimidines andprodrugs thereof for reducing tumor burden without inhibiting kinases.As such, this lessens their potential for mitochondrial toxicity byinhibition of pol gamma or other human polymerases.

In one aspect, the present invention provides for a nucleobase analoguecompound of formula (I) or prodrug thereof:

wherein X is S or NH and Y is H, Br or I.

In another aspect, the present invention provides for a method ofinducing apoptosis and/or inhibiting the growth of cancer cells, themethod comprising administering a therapeutically effective amount of ahalogenated pyrrolopyrimidine, thienopyrimidine or prodrug thereofhaving the following formula:

wherein X is S or NH and Y is H, Br or I.

In yet another aspect, the present invention provides for a prodrug ofnucleobase analogue compound of formula (II):

wherein X is S or NH;

R is H, Br, NH or I;

Y is I, Cl, OCH₃, O-benzyl, F, NH₂ or C₂H₃N₃; and

Z is Cl, O-benzyl, F or OCH₃.

In a further aspect, the present invention provides for the use of acompound of formula formula (III) in the manufacture of a medicament forthe treatment of cancer having the following structure:

wherein R¹ and R² are shown below in different configurations andstructures:5. R¹═R^(2=H)6. R¹═H, R²═Br7. R¹═H, R²═I8. R¹=BOM, R²═I9. R¹=Tos, R²═I.wherein BOM is benzyloxymethyl and Tos is p-toluenesulfonyl group

Other protective groups useful in the protection of an amide groupinclude, but not limited to, tert-Butoxy carbamate (Boc),9-Fluorenylmethoxycarbonyl (Fmoc), Methoxymethyl acetal (Mom), Benzyl,or Acetyl protection. Removal of the protective group can be effected byan enzymatic reaction, hydrolysis or a catalyzed reaction.

Structures of additional pyrrolo[3,2-d]pyrimidine prodrugs are shownbelow:

In another aspect, the present invention provides for a pharmaceuticalcomposition comprising a compound of formula (III) and apharmaceutically acceptable carrier.

In a further aspect, the present invention provides method of treatingcancer, comprising: providing to a subject a prodrug of a halogenatedpyrrolopyrimidine or thienopyrimidine compound in an amount that yieldsa therapeutically-effective amount the halogenated pyrrolopyrimidine orthienopyrimidine compound.

In a still further aspect, the present disclosure provides methods ofinhibiting cancer proliferation activity and the use thereof for cancertreatment in an individual, the methods involving administering to theindividual a prodrug having the following structure

that is converted in the individual to the following compound

Notably, the prodrug is converted, by some chemical or physiologicalprocess in the individual to the halogenated pyrrolopyrimidine compound.

In yet another aspect, the present invention provides for contacting acell in an animal, such as a human cell, with at least one of thenucleobase analogues or prodrug thereof provided herein.

Other aspects, features and embodiments of the invention will be morefully apparent from the ensuing disclosure and appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 (A) is a scheme for synthesis of2,4-Dichlorothieno[3,2-d]pyrimidine (1), (B) scheme for synthesis ofBromo-2,4-dichlorothieno[3,2-d]pyrimidine (2).

FIG. 2 is a scheme for the synthesis halogenatedpyrrolo[3,2-d]pyrimidine (19).

FIG. 3 (A) is a scheme for the synthesis of 7-iodo-2,4-dichloropyrrolo[3,2-d]pyrimidine (20) (B) is a scheme for the synthesis of7-iodo-2,4-bis-benzyloxy-5H-pyrrolo[3,2-d]pyrimidine (16).

FIG. 4 shows the results of anti-cancer activity of lead compounds andthe growth percent of representative cancer cell line from the NCI60cell line (1 dose).

FIG. 5 (A) shows levels of apoptosis of control (i) and compound 1(i);(B) shows apoptosis of compound 2 (ii) and compound 19 (iv).

FIG. 6 shows in vivo activity and toxicity of lead compounds 1 and 2,(A) depicts the mean tumor responsiveness in an in vivo activity screen;(B) shows the percent body weight loss versus time for groups of micereceiving IP doses of compound for 5 consecution days.

FIG. 7 shows the cell cycle distributions of L1210 cells following 0,24, or 48 hours in the presence of a. 0.32 μM compound 1, b. 5.4 μMcompound 2, and analyzed by flow cytometry of fixed, propidiumiodide-stained cells. A minimum of 3000 cells were analyzed per cellpopulation. Each bar represents the mean±SD across 4 independent cellsamples.

FIG. 8 shows the Cell cycle analyses of MDA-MB-231 cells followingtreatment with compounds 1 and 2. Cell cycle distributions of MDA-MB-231cells after 48 hours in the presence of equitoxic (IC80) doses of (a)compound 1 (15 μM) or (b) compound 2 (8 μM) analyzed by flow cytometryof fixed, propidium iodide-stained cells and compared to vehiclecontrols (DMSO). A minimum of 3000 cells were analyzed per cellpopulation. Each bar represents the mean±SD across 4 independent cellsamples.

FIG. 9 shows the cell cycle distributions of L1210 cells following 48hours in the presence of a. 1.75 μM compound 20, b. 15 μM compound 19,and analyzed by flow cytometry of fixed, propidium iodide-stained cells.A minimum of 3000 cells were analyzed per cell population. Each barrepresents the mean±SD across 4 independent cell samples.

FIG. 10 shows the results of anti-cancer activity of compound 1 and thegrowth percent of representative cancer cell line from the NCI60 cellline (1 dose).

FIG. 11 shows the results of anti-cancer activity of lead compound 2 andthe growth percent of representative cancer cell line from the NCI60cell line (1 dose).

FIG. 12 shows the results of anti-cancer activity of lead compound 19and the growth percent of representative cancer cell line from the NCI60cell line (1 dose).

FIG. 13 shows the results of anti-cancer activity of lead compounds 1and 2 and the growth percent of representative cancer cell line from theNCI60 cell line (5 dose).

FIG. 14 shows the in vitro conversion of compound 9 to compound 7. Datais reported in analyte counts to control for variation between standardcurves for the two compounds.

DETAILED DESCRIPTION OF THE INVENTION

The thieno[3,2-d]pyrimidine compounds are known because of their closeresemblance to the purines, arguably the most biologically significantclass of bicyclic heterocyclic compounds [10-15]. Indeed,thienopyrimidine derivatives have been extensively studies by severalindependent groups and several molecules have been identified withantiviral, antimicrobial or kinase inhibition activities. In addition,thienopyrimidine scaffolds carrying an aromatic ring substituent possessenhanced anti-tumor activity, which was proved by testing them againstcolorectal cancer cell lines. However, such cancer proliferationinhibitory activity was fairly modest, with an IC₅₀ in the high uM range[16-25].

The present inventors have has worked on the generation of thiophene“extended” pyrimidine nucleosides [26, 27] but such nucleosides showedonly modest results for anti-cancer activities [28].

In the present invention it has been found that halogenatedpyrrolopyrimidines and thienopyrimidines compounds are pharmacologicallyuseful scaffolds that manifest anti-tumor activity against cancer by atleast inducing apoptosis. Importantly, it has been found that a halogenat the C4 and/or C7 exhibits increase potency. In preferred embodiments,Y at C4 is chlorine or O-Benzyl, R at C7 is I and X is S or NH.

Definitions

The term “treat” or “treatment” as used herein refers to boththerapeutic treatment and prophylactic or preventative measures, whereinthe compound is to slow down (lessen) an undesired pathological changeor disorder, such as the development or spread of cancer. For purpose ofthis invention, beneficial or desired clinical results include, but arenot limited to, alleviation of symptoms, diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. For example, “treatment” can include aqualitative or quantitative reduction (e.g., by at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) in the tumor or metastasessize or reduce or prevent metastatic growth. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment.

The term “prodrug” as used herein is meant to refer to any compound thatundergoes biotransformation before exhibiting its pharmacologicaleffects. Thus, this includes a compound that is a drug precursor which,following administration to a subject, releases the drug in vivo viasome chemical or physiological process such that the prodrug isconverted into a product that is toxic to cancer cells. An amount of theprodrug that yields a therapeutically-effective amount of the activedrug after administration is an amount of that is effective toameliorate or minimize the clinical impairment or symptoms of thecancer, in either a single dose or multiple doses.

The phrase “therapeutically effective amount” as used herein refers toan amount of a compound of the present invention that (i) treats orprevents the particular disease, condition, or disorder, (ii)attenuates, ameliorates, or eliminates one or more symptoms of theparticular disease, condition, or disorder, or (iii) prevents or delaysthe onset of one or more symptoms of the particular disease, condition,or disorder described herein. In the case of cancer, the therapeuticallyeffective amount of the drug may be reduce the number of cancer cells;reduce the tumor size; inhibit (i.e., slow to some extent and preferablystop) cancer cell infiltration into peripheral organs; inhibit (i.e.,slow to some extent and preferably stop) tumor metastasis; inhibit, tosome extent, tumor growth; and/or relieve to some extent one or more ofthe symptoms associated with the cancer. For cancer therapy, efficacycan be measured, for example, by assessing the time to diseaseprogression (TTP) and/or determining the response rate (RR).

The terms “cancer” or “cancerous” as used herein refer to or describethe physiological condition in mammals that is typically characterizedby unregulated cell growth. A “tumor” comprises one or more cancerouscells. Examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include squamous cell cancer (e.g.,epithelial squamous cell cancer), lung cancer including small-cell lungcancer, non-small cell lung cancer (“NSCLC”), adenocarcinoma of the lungand squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatome, breastcancer, colon cancer, rectal cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, as well as head and neck cancer.

The term “salt” as used herein includes base addition, acid addition andquaternary salts. Compounds of the invention which are acidic can formsalts, including pharmaceutically or veterinarily acceptable salts, withbases such as alkali metal hydroxides, e.g. sodium and potassiumhydroxides; alkaline earth metal hydroxides e.g. calcium, barium andmagnesium hydroxides; with organic bases e.g. N-ethyl piperidine,dibenzylamine and the like. Those compounds which are basic can formsalts, including pharmaceutically or veterinarily acceptable salts withinorganic acids, e.g. with hydrohalic acids such as hydrochloric orhydrobromic acids, sulphuric acid, nitric acid or phosphoric acid andthe like, and with organic acids e.g. with acetic, tartaric, succinic,fumaric, maleic, malic, salicylic, citric, methanesulphonic andp-toluene sulphonic acids and the like.

The term ‘solvate’ as used herein refers to a molecular complexcomprising the compound of the invention and a stoichiometric amount ofone or more pharmaceutically acceptable solvent molecules, for example,ethanol. The term ‘hydrate’ is employed when said solvent is water.

Compounds with which the invention is concerned may exist in one or morestereoisomeric form because of the presence of asymmetric atoms orrotational restrictions and can exist as a number of stereoisomers withR or S stereochemistry at each chiral centre or as atropisomeres with Ror S stereochemistry at each chiral axis. The invention includes allsuch enantiomers and diastereoisomers and mixtures thereof.

While it may be possible for compounds of the present invention to beadministered as the raw chemical, it is preferable to present them as apharmaceutical composition. According to a further aspect, the presentinvention provides a pharmaceutical composition comprising a compound ora mixture of compounds of Formula (I) and/or Formula (II) or apharmaceutically acceptable salt, solvate or hydrate thereof, togetherwith one or more pharmaceutical carrier, excipient or additive andoptionally one or more other therapeutic ingredients. The carrier(s)must be “acceptable” in the sense of being compatible with the otheringredients of the formulation and not deleterious to the recipientthereof. The term “pharmaceutically acceptable carrier” includesvehicles and diluents.

To prepare the pharmaceutical compositions, a therapeutically effectiveamount of one or more of the halogenated pyrrolopyrimidines andthienopyrimidines according to the present invention may be admixed witha pharmaceutically acceptable carrier according to conventionalpharmaceutical compounding techniques to produce a dose. A carrier maytake a wide variety of forms depending on the form of preparationdesired for administration, e.g., oral, topical or parenteral, includinggels, creams ointments, lotions and time released implantablepreparations, among numerous others. In preparing pharmaceuticalcompositions in oral dosage form, any of the usual pharmaceutical mediamay be used. Thus, for liquid oral preparations such as suspensions,elixirs and solutions, suitable carriers and additives including water,glycols, oils, alcohols, flavoring agents, preservatives, coloringagents and the like may be used. For solid oral preparations such aspowders, tablets, capsules, and for solid preparations such assuppositories, suitable carriers and additives including starches, sugarcarriers, such as dextrose, mannitol, lactose and related carriers,diluents, granulating agents, lubricants, binders, disintegrating agentsand the like may be used. If desired, the tablets or capsules may beenteric-coated or sustained release by standard techniques.

In one embodiment, the compositions are prepared with carriers that willprotect the active compound(s) against rapid elimination from the body,such as a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art.

The pharmaceutically acceptable carrier may take a wide variety offorms, depending on the route desired for administration, for example,oral or parenteral (including intravenous). Carriers such as starches,sugars, microcrystalline cellulose, diluents, granulating agents,lubricants, binders and disintegrating agents may be used in the case oforal solid preparations such as powders, capsules and caplets, with thesolid oral preparation being preferred over the liquid preparations.Preferred solid oral preparations are tablets or capsules, because oftheir ease of administration. If desired, tablets may be coated bystandard aqueous or nonaqueous techniques. Oral and parenteral sustainedrelease dosage forms may also be used.

Liposomal suspensions may also be pharmaceutically acceptable carriers.These may be prepared according to methods known to those skilled in theart. For example, liposomal formulations may be prepared by dissolvingappropriate lipid(s) in an inorganic solvent that is then evaporated,leaving behind a thin film of dried lipid on the surface of thecontainer. An aqueous solution of the active compound is then introducedinto the container. The container is then swirled by hand to free lipidmaterial from the sides of the container and to disperse lipidaggregates, thereby forming the liposomal suspension. Other methods ofpreparation well known by those of ordinary skill may also be used inthis aspect of the present invention.

In an embodiment, the composition of the present invention enablessustained, continuous delivery of a compound of Formula (I) or Formula(II) a pharmaceutically acceptable salt, solvate or hydrate thereof, totissues adjacent to or distant from an administration site. Thebiologically-active agent is capable of providing a local or systemicbiological, physiological or therapeutic effect. For example, a compoundof Formula (I) or Formula (II) a pharmaceutically acceptable salt,solvate or hydrate thereof, can act to kill cancer cells or to controlor suppress tumor growth or metastasis, among other functions.

Pharmaceutical formulations based upon halogenated pyrrolopyrimidine andthienopyrimidine compounds of the present invention comprise at leastone of the compounds of Formula (I) or Formula (II) or a prodrugthereof, in a therapeutically effective amount for treating canceroptionally in combination with a pharmaceutically acceptable additive,carrier and/or excipient. One of ordinary skill in the art willrecognize that a therapeutically effective amount of one of morecompounds according to the present invention will vary with thecondition to be treated, its severity, the treatment regimen to beemployed, the pharmacokinetics of the agent used, as well as the patient(animal or human) treated.

The formulations of the present invention include those suitable fororal, parenteral (including subcutaneous, intradermal, intramuscular,intravenous, intratumoral and intraarticular), rectal and topical(including dermal, buccal, sublingual and intraocular) administration,as well as those for administration by inhalation. The most suitableroute may depend upon the condition and disorder of the recipient.Exemplary formulations are well known to those skilled in the art, andgeneral methods for preparing them are found in any standard pharmacyschool textbook, for example, Remington: THE SCIENCE AND PRACTICE OFPHARMACY, 21st Ed., Lippincott. The formulations of the presentinvention may conveniently be presented in unit dosage form and may beprepared by any of the methods well known in the art of pharmacy. Allmethods include the step of bringing into association a compound or apharmaceutically acceptable salt or solvate thereof (“activeingredient”) with the carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both and then, ifnecessary, shaping the product into the desired formulation.

The concentration of active compound of the present invention, i.e., atleast one of the compounds of Formula (I) or (II) or a prodrug thereof,in the drug composition will depend on absorption, distribution,inactivation, and excretion rates of the drug as well as other factorsknown to those of skill in the art. It is to be noted that dosage valueswill also vary with the severity of the condition to be alleviated. Thecomposition may be administered at once, or may be divided into a numberof smaller doses to be administered at varying intervals of time.

Oral compositions will generally include an inert diluent or an ediblecarrier. They may be enclosed in gelatin-capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches and the like can contain any ofthe following non-limiting ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a dispersing agent suchas alginic acid or corn starch; a lubricant such as magnesium stearate;a glidant such as colloidal silicon dioxide; a sweetening agent such assucrose or saccharin; or a flavoring agent such as peppermint, methylsalicylate, or fruit flavoring. When the dosage unit form is a capsule,it can contain, in addition to any of the above, a liquid carrier suchas a fatty oil. In addition, dosage unit forms can contain various othermaterials which modify the physical form of the dosage unit, forexample, coatings of sugar, shellac, or enteric agents.

The active compound or prodrug thereof may also be administered as acomponent of an elixir, suspension, syrup, wafer or the like. A syrupmay contain, in addition to the active compound or prodrug thereof,sucrose or fructose as a sweetening agent and certain preservatives,dyes and colorings and flavors.

In certain embodiments of the present invention, the halogenatedpyrrolopyrimidines, thienopyrimidines or prodrugs thereof are formulatedas admixture with a pharmaceutically acceptable carrier, excipient oradditive. The pharmaceutical composition may be administered via atopical, parenteral, intravenous, intramuscular, transdermal, buccal,subcutaneous, suppository or other route, including an eye or ocularroute. Intravenous and intramuscular formulations are generallyadministered in sterile saline. Of course, one of ordinary skill in theart may modify the formulations within the teachings of thespecification to provide numerous formulations for a particular route ofadministration without rendering the pharmaceutical compositionsunstable or compromising their therapeutic activity.

Pharmaceutical compositions containing any of the compounds of Formula(I), Formula (II) or a prodrug thereof, may be conveniently presented inunit dosage form and prepared by any of the methods well known in theart of pharmacy. In general, a therapeutically effective amount of thepresent preferred compound in dosage form usually ranges from slightlyless than about 0.025 mg/kg to about 5 g/kg of body weight, and incertain embodiments about 2.5 mg/kg to about 750 mg/kg of body weight orabout 5 mg/kg to about 250 mg/kg of body weight of the patient,depending upon the compound used, the condition being treated and theroute of administration, although exceptions to this dosage range may becontemplated by the present invention.

In an embodiment, the present invention is also directed to methods forthe treatment of tumors and/or cancer comprising administering aneffective amount of one or more halogenated pyrrolopyrimidines,thienopyrimidines or prodrugs thereof to a patient in need of suchtherapy. For example, the present invention contemplates methods oftreating various cancers and complications thereof. More particularly,the present invention relates to methods for inhibiting the growth ofbenign and malignant cancer, including a malignant tumor or cancercomprising exposing the tumor to an inhibitory or therapeuticallyeffective amount or concentration of at least one of the halogenatedpyrrolopyrimidines and thienopyrimidines or prodrugs thereof. Treatmentof internal malignancies such as eye or ocular cancer, rectal cancer,colon cancer, cervical cancer, prostate cancer, breast cancer, livercancer and bladder cancer, and age-related cancer among numerous othersare contemplated by the present invention.

Accordingly, the compounds and/or compositions of the present inventionare useful for treating animals, and in particular, mammals, includinghumans, as patients. Thus, humans and other animals, and in particular,mammals, suffering from hyperproliferative disorders, and in particular,cancer, or other diseases as disclosed herein, can be treated byadministering to the patient an effective amount of one or more of thehalogenated pyrrolopyrimidines, thienopyrimidines or prodrugs accordingto the present invention, optionally in a pharmaceutically acceptablecarrier or diluent, either alone, or in combination with other knownpharmaceutical agents (depending upon the disease to be treated).Treatment according to the present invention can also be byadministration of the compounds or prodrugs of the present invention inconjunction with other conventional cancer therapies, such as radiationtreatment or surgery or administration of other anti-cancer agents.

The cancer may be a solid tumor, metastatic cancer, or non-metastaticcancer. In certain embodiments, the cancer may originate in the bladder,blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum,small intestine, large intestine, colon, rectum, anus, gum, head,kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach,testis, tongue, or uterus. In certain embodiments, the cancer is ovariancancer. In particular aspects, the cancer may be a chemo-resistantcancer, i.e., refractive forms of cancer.

The methods and compositions of the present invention further providecombination therapies which can enhance the therapeutic or protectiveeffect of the compounds of the present invention, and/or increase thetherapeutic effect of another anti-cancer or anti-hyperproliferativetherapy. Therapeutic and prophylactic methods and compositions can beprovided in a combined amount effective to achieve the desired effect,such as the killing of a cancer cell and/or the inhibition of cellularhyperproliferation. Further, a tissue, tumor, or cell can be contactedwith the compounds or compositions of the present invention and one ormore additional anti-cancer treatment. For example, an additionalanticancer treatment may include a chemotherapeutic agent, radiotherapy,surgical therapy, or immunotherapy.

Examples of chemotherapeutic agents include alkylating agents such asthiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylol melamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicinomegall; dynemicin, including dynemicin A; bisphosphonates, such asclodronate; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores, aclacinomysins,actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogues such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogues such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonicacid; triaziquone; 2,2′,2″-trichlorotriethylamine, trichothecenes(especially T-2 toxin, verracurin A, roridin A and anguidine); urethan;vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide;thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumcoordination complexes such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-I1); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoids such as retinoic acid; capecitabine; cisplatin (CDDP),carboplatin, procarbazine, mechlorethamine, cyclophosphamide,camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea,dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin,mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptorbinding agents, taxol, paclitaxel, docetaxel, gemcitabien, navelbine,farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil,vincristine, vinblastine and methotrexate and pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

Within a single day (24-hour period), the patient may be given one ormultiple administrations of the halogenated pyrrolopyrimidines,thienopyrimidines or prodrugs thereof. The course of treatment may last1, 2, 3, 4, 5, 6, 7 days, and/or 1, 2, 3, 4, 5 weeks, and/or 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 months or more, depending on the condition ofthe patient, such as their prognosis, strength, health, etc. Moreover,after a course of treatment, it is contemplated that there is a periodof time at which no anti-cancer treatment is administered. This timeperiod may last 1, 2, 3, 4, 5, 6, 7 days, and/or 1, 2, 3, 4, 5 weeks,and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more.

EXAMPLES

The following examples are intended to further illustrate certainpreferred embodiments of the invention and are not limiting in nature.Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific substances and procedures described herein.

Preparative Examples

Experimental.

All chemicals and reagents listed in this section were purchased throughcommercially available sources unless otherwise noted. All reactions runin CH₂Cl₂, CH₃CN, and THF were obtained from a solvent purificationsystem (SPS, Model: mBraun Labmaster 130). All reactions run inanhydrous DMF, MeOH and pyridine were obtained from Sigma-Aldrich orAcros Organics. All ¹H and ¹³C NMR spectra were obtained from a JEOL ECX400 MHz NMR. All ¹H and ¹³C NMR spectra were referenced to internaltetramethylsilane (TMS) at 0.0 ppm. The spin multiplicities areindicated by the symbols s (singlet), d (doublet), dd (doublet ofdoublets), t (triplet), q (quartet), m (multiplet), and br (broad). AllNMR solvents were obtained from Cambridge Isotope Laboratories. Allreactions were monitored by thin layer chromatography (TLC) on 0.25 mmprecoated glass plates. All column chromatography was run on 32-63 usilica gel obtained from Dynamic Adsorptions Inc. (Norcross, Ga., USA).Melting points are uncorrected. Yields refer to chromatographically andspectroscopically (¹H and ¹³C NMR) homogeneous materials. All massspectra (MS) were recorded and obtained from the University of MarylandBaltimore County Mass Spectrometry Facility and Johns Hopkins MassSpectrometry Facility. The FAB mass spectra were obtained using doublefocusing magnetic sector mass spectrometer equipped with a Cs ion gunand fourier transform ion cyclotron resonance equipped with ESI source.

Example 1 Synthesis of 2,4-Dichlorothieno[3,2-d]pyrimidine (1) (FIG. 1A)Thieno[3,2-d]pyrimidin-2,4(1H,3H)-dione (4)

In a dry flask methyl-3-amino-2-thiophene carboxylate 3 (5.00 g, 31.80mmol) was dissolved in acetic acid (100 mL) to obtain a yellow solutionto which potassium cyanate (10.31 g, 127.30 mmol) dissolved in water (80mL) was added dropwise over 3 h. The resultant suspension was stirredovernight (16 hr) at which point the suspension was filtered. The whitesolid residue was dissolved in 2N NaOH (80 mL) by warming to 70° C. Theclear solution was then acidified by AcOH (pH 4-5), the resulting whiteprecipitate was filtered, washed with water then acetone and dried toobtain 4 as a white solid (3.80 g, 22.60 mmol, 71%).

2,4-Dichlorothieno[3,2-d]pyrimidine (1)

In a dry flask, thieno[3,2-d]pyrimidin-2,4(1H,3H)-dione 4 (4.00 g, 23.78mmol) was refluxed in freshly distilled POCl₃ (50 mL) under nitrogenovernight (16 h) at which point the POCl₃ was evaporated and the residueextracted with CH₂Cl₂ (50 mL). The organic layer was washed withsaturated NaHCO₃ solution (50 mL), brine (50 mL), dried over MgSO₄ andconcentrated. The residue was crystallized from EtOAc to obtain 1 as apale green-yellow solid (4.00 g, 19.50 mmol, 82%). Mp: 135-137° C. ¹HNMR (400 MHz, CDCl₃): δ 7.55 (d, J=5.0 Hz), 8.12 (d, J=5.5 Hz). ¹³C NMR(100 MHz, CDCl₃): δ 124.6, 129.4, 139.3, 155.8, 156.32, 163.5. FAB-MSm/z for C₆H₂Cl₂N₂S calculated [M+H]⁺ 204.9388, found 204.9400 (2×³⁵Cl),206.9366 (³⁵Cl ³⁷Cl).

Example 2 Synthesis of Bromo-2,4-dichlorothieno[3,2-d]pyrimidine (FIG.1B) 7-Bromo-thieno[3,2-d]pyrimidin-2,4 (1H, 3H)-dione (22)

To 100 mL glass tube containing thieno[3,2-d]pyrimidin-2,4-dione 4 (5.88g, 34.86 mmol), AcOH (60 mL) and bromine (3.6 mL, 69.72 mmol) were addedand the tube capped. The sealed tube was stirred in a preheated oil bathat 90° C. for 24 h. An additional portion of bromine (3.6 mL, 69.72mmol) was added to the sealed tube and mixture stirred for another 24 hat 90° C. The AcOH was evaporated to obtain a solid residue to whichwater was added (200 mL) and the suspension filtered and residue washedrepeatedly with water and dried under vacuum to obtain 22 as anoff-white solid (8.07 g, 32.67 mmol, 94%). Mp 251.0-252.9° C. ¹H NMR(400 MHz, DMSO-d₆): δ 8.22 (s, ¹H), 11.40 (br s, ¹H, NH), 11.58 (br s,1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δ 99.6, 112.1, 133.4, 145.1, 152.1,159.0. FAB-MS m/z for C₆H₃BrN₂O₂S calculated [M+H]⁺ 246.9171, found246.9175 (⁷⁹Br), 248.9161 (⁸¹Br).

7-Bromo-2,4-dichlorothieno[3,2-d]pyrimidine (2)

To a round bottom flask containing7-bromothieno[3,2-d]pyrimidin-2,4-dione 22 (4.07 g, 16.47 mmol), DMAP(8.38 g, 68.76 mmol) and freshly distilled POCl₃ were added and thesuspension stirred at 105-110° C. for 2 h under nitrogen. The POCl₃ wasevaporated and the residue extracted with CH₂Cl₂ (300 mL). The organiclayer was washed with aq. NaHCO₃(300 mL), brine (200 mL) and dried overMgSO₄. The dried organic layer was concentrated, loaded on silica andthe product purified using column chromatography eluting with 19:1hexanes:EtOAc to obtain 2 as a white solid (3.41 g, 12.00 mmol, 73%).R_(f) 0.5 in 9:1 hexanes/EtOAc. Mp 180.2-183.0° C. ¹H NMR (400 MHz,CDCl₃): δ 8.13 (s, ¹H). ¹³C NMR (100 MHz, CDCl₃): δ 160.0, 158.0, 156.0,135.0, 128.0, 109.5. FAB-MS m/z for C₆HBrCl₂N₂S calculated [M+H]⁺282.8493, found 282.8495 (2×³⁵Cl, ⁷⁹Br), 284.8468 (2×³⁵Cl, ⁸¹Br),286.8443 (³⁵Cl, ³⁷Cl, ⁸¹Br), 288.8419 (2×³⁷Cl, ⁸¹Br).

Example 3 Synthesis of Halogenated pyrrolo[3,2-d]pyrimidine (FIG. 2)

Pyrrolo[3,2-d]pyrimidin-2,4-dione (18) To a stirred slurry of(E)-6-(2-(dimethylamino)vinyl)-5-nitropyrimidin-2,4-dione ^(35,36) (1 g,4.4 mol) in fresh glacial AcOH (50 mL), Zinc dust (stabilized) (2 g) wasadded in two lots of 1 g with an interval of 1 h. Upon overnightstirring the yellow slurry changed to a pale yellow to off-white slurrywhich was filtered and the filtrate concentrated in vacuo to obtainbrown syrup. The product was precipitated from the brown syrup usingethanol to obtain as white solid (0.6 g, 89.8%). The spectral dataagrees with reported data. ¹H NMR (400 MHz, DMSO-d₆): δ 5.82-5.83 (t,J=2.28 Hz), 7.12-7.13 (t, J=2.72 Hz, J=2.96 Hz), 10.57 (s, 1H), 10.74(s, 1H), 11.82 (s, 1H). ¹³C NMR (400 MHz, DMSO-d₆): δ 96.5, 110.9,127.4, 135.1, 152.0, 156.3.

2,4-Dichloropyrrolo[3,2-d]pyrimidine (19)

To pyrrolo[3,2-d]pyrimidin-2,4-dione 18 (2.00 g, 13.2 mmol), 1N NaOH (15mL), and 0.60 g NaOH in 15 mL H₂O was added and the mixture stirred at40° C. until a solution was formed. The solution was then cooled to roomtemperature (21-25° C.) and then placed in an ice bath to obtain thickslurry. The slurry was then filtered to obtain a pale yellow solid. Thesolid was dissolved in 1N NaOH (15 mL), and heated to 40° C. to obtain aclear solution that upon cooling provided white crystals. The crystalswere washed with MeOH (20 mL) and acetone (20 mL), and then dried undervacuum. The dry solids were taken in phenylphosphonic dichloride (10 mL)and heated to 170-175° C. for 5 h during which the reaction mixturebecame a brown-black solution. After 5 h the hot reaction mixture waspoured onto ice, extracted with EtOAc (200 mL) and the organic layerwashed with sat. NaHCO₃ solution (3×100 mL) till all effervescencesubsided. The organic layer was then washed with brine and dried overMgSO₄. The organic layer was concentrated in vacuo and loaded ontosilica. The product was purified using column chromatography elutingwith 9:1 then 3:1 hexanes/EtOAc to obtain compound 18 as an off-whitesolid (1.50 g, 7.9 mmol, 60%). Rf 0.5 in 3:1 hexanes/EtOAc. Mp228.3-232.0° C. 1H NMR (400 MHz, DMSO-d6): δ 6.71 (d, J=3.2 Hz), 8.09(d, J=2.8 Hz), 12.75 (s, ¹H, NH). ¹³C NMR (100 MHz, DMSO-d6): δ 103.2,124.3, 138.0, 143.5, 149.6, 153.9. ESI-MS m/z for C₆H₃Cl₂N₃ calculated[M+H]+ 187.9776, found 187.9777.

Example 4 Synthesis of 7-iodo-2,4-dichloro pyrrolo[3,2-d]pyrimidine (20)FIG. 3A

To a solution of 2,4-dichloro pyrrolo[3,2-d]pyrimidine 19 (100 mg, 0.53mmol) in anhydrous THF (5 mL), NIS (144 mg, 0.64 mmol) was added underN₂ atmosphere and stirred for 2 h after which TLC indicated consumptionof 19. The solvent was removed in vacuo and the residue dissolved inEtOAc. The organic phase was washed with aq. solution of Na₂S₂O₃followed by water, brine and then dried over MgSO₄. The organic layerwas concentrated in vacuo and loaded on silica. The product 20 waspurified using column chromatography eluting with 9:1 hexanes/EtOAc toobtain product as off-white solid (90 mg, 54%). R_(f) 0.55 in 3:1hexanes/EtOAc. Mp decomposed from 160-230° C. ¹H NMR (400 MHz, DMSO-d₆):δ 8.29 (s, ¹H), 13.19 (s, NH). ¹³C NMR (400 MHz, DMSO-d₆): δ 58.2,124.4, 140.9, 143.5, 149.8, 153.5. ESI-MS m/z for calculated [M+H]⁺313.8743, found 313.8740.

Example 5 Synthesis of7-iodo-2,4-bis-benzyloxy-5H-pyrrolo[3,2-d]pyrimidine (16) FIG. 3B

2,4-Bis-benzyloxy-5-nitro-6-dimethylaminovinyl pyrimidine (14)

To a solution of 2,4-Bis-O-Benzyl-6-methyl-5-nitro pyrimidine 13 [37](2.3 g, 6.5 mmol) in DMF (20 mL), DMF-dimethyl acetal (1.74 mL, 13 mmol)was added at room temperature under N₂ atmosphere. The reaction waslowered in a preheated oil bath at 60-65° C. and stirred overnight uponwhich TLC indicated absence of starting material. The solvents wereremoved and the residue loaded on silica. The product was purified usingcolumn chromatography eluting with 9:1 hexanes/EtOAc to obtain product14 as orange-yellow solid (2 g, 75%). R_(f) 0.5 in 3:1 hexanes/EtOAc. ¹HNMR (400 MHz, CDCl₃): δ 2.87-2.94 (br d, 6H), 5.33-5.36 (d, 1H, J=12.36Hz), 5.38 (s, 2H), 5.44 (s, 2H), 7.31-7.41 (m, 10H), 7.98-8.01 (d, 1H,J=12.36 Hz). ¹³C NMR (400 MHz, CDCl₃): δ 68.9, 69.5, 87.9, 127.5, 128.0,128.1, 128.3 128.5, 135.8, 136.6, 151.8, 160.7, 161.6, 163.5. FAB-MS forC₂₂H₂₂N₄O₄ calculated [M+H]⁺ 407.1714, found 407.1717.

2,4-Bis-benzyloxy-5H-pyrrolo[3,2-d]pyrimidine (15)

To a suspension of 2,4-Bis-O-Benzyl-5-nitro-6-β-dimethylaminovinylpyrimidine 14 (2 g, 4.9 mmol) in AcOH (40 ml), Zn (4 g) was added in lotof 2 g with an interval of 4 hrs. The reaction mixture was stirredovernight at room temperature during which a dark yellow suspensionbecame pale yellow suspension. The reaction mixture was filtered and thefiltrate concentrated in vacuo to obtain syrup which was dissolved inCH₂Cl₂ then washed with saturated aq. NaHCO₃ followed by brine. Theorganic phase was dried over MgSO₄ and loaded on silica. The product 15was purified using column chromatography eluting with 4:1 and 1:1hexanes/EtOAc to obtain product as pale-yellow solid (1.45 g, 90%).R_(f) 0.3 in 1:3 hexanes/EtOAc. ¹H NMR (400 MHz, CDCl₃): δ 5.47 (s, 2H),5.54 (s, ¹H), 6.50-6.51 (dd, 1H, J=1.84, 2.28), 7.29-7.38 (m, 7H),7.43-7.45 (m, 2H), 7.52-7.53 (m, 2H), 8.41 (br s, 1H, NH). ¹³C NMR (400MHz, CDCl₃): δ 68.3, 69.0, 102.6, 111.9, 127.8, 128.3, 128.4, 128.5,128.6, 128.7, 128.8, 136.1, 137.3, 151.8, 156.79, 159.7. FAB-MS forC₂₀H₁₇N₃O₂ calculated [M+H]⁺ 332.1394, found 332.1398.

7-iodo-2,4-bis-benzyloxy-5H-pyrrolo[3,2-d]pyrimidine (16)

To a stirred solution of 2,4-bis-O-benzyl-5H-pyrrolo[3,2-d]pyrimidine 15(1.43 g, 4.3 mmol) in anhydrous CH₂Cl₂ (15 mL) under N₂, NIS (1.069 g,4.7 mmol) was added at which point the reaction mixture turned from pinkto orange. The mixture was stirred overnight until the TLC indicated theabsence of starting material. The reaction mixture was washed withaqueous Na₂S₂O₃ (15 mL) followed by brine (15 mL). The organic layer wasdried over MgSO₄, loaded onto silica and purified using columnchromatography eluting with 4:1 then 1:1 hexanes/EtOAc to obtaincompound 16 as a pale-yellow solid (1.77 g, 3.88 mmol, 90%). R_(f) 0.4in 1:3 hexanes/EtOAc. Mp 157.8-158.4° C. ¹H NMR (400 MHz, CDCl₃): δ 5.53(s, 4H), 7.32-7.41 (m, 9H), 7.55-7.57 (m, 2H), 8.71 (br s, 1H, NH). ¹³CNMR: 57.3, 68.7, 69.3, 111.9, 127.9, 128.3, 128.6, 128.66, 128.7, 128.9,132.4, 135.8, 137.2, 152.0, 156.9, 160.1. FAB-MS m/z for C₂₀H₁₆IN₃O₂calculated [M+H]⁺ 458.0360, found 458.0357.

Example 6

Lead compounds 1, 2 and 19 were evaluated for anti-cancer activity at nMto uM concentrations in cellular screens. All three compounds exhibitedselective activity against melanoma, breast, colon, and renal cancers(NCI60 cell line) but with little or no activity against non-small celllung and CNS cancers, as shown in FIG. 4. Two leads, that beingcompounds 1 and 2, exhibited inhibition by 50% in a 5 dose NCI 60 cellline screen (G150). Although their exact mechanism of action has notbeen fully elucidated, it is believed that these compounds do notinhibit DNA polymerase or kinases (see Example 12 hereinbelow).

FIGS. 5 A and B contains apoptosis analysis (annexin V staining) in theL1210 leukemia cell line relating to compounds 1 (ii), 2 (iii) and 19(iv) relative to a control (i). After incubation with 1 μM compound (1)for 48 h, 60% of the cells were observed undergoing apoptotic celldeath, with the vast majority of these in early apoptosis (FIG. 5A(ii)). Cell death consistent with apoptosis was also evident in L1210cells treated with 5.4 μM solution of compound 2 where 55% of cells wereAnnexin V positive (FIG. 5B (iii), although a larger proportion of these(40%) were consistent with late apoptosis. Compound 19 was incubated for48 hours in the amount of 1 μM and the compound exhibited 85% apoptosiswherein the majority was early apoptosis. These results of apoptosisassays indicate that compounds 1, 2 and 19 can induce cell death by anapoptotic-like pathway, but in a manner that does not require arrest ata specific stage of the cell cycle.

Example 7

The selectivity of these pyrimidine analogues for some cancer cell linessuggests that safe and effective doses can be achieved. The histogram inFIG. 6A shows the mean tumor responsiveness ±the SEM in an in vivoactivity screen using intra-tumor injection (5 days on/2 days off) in anamount of 0.01 mg/100 mm³ tumor for 21 days. Notably the amount ofdosage was based on the size of the tumor because the drug wasadministered directly into the tumor.

The asterisk denotes p>0.05 when compared to the vehicle only (n=6-10mice). Melanoma is viewed as resistant to nucleoside analogues, sincetraditional FDA-approved nucleosides have minimal effects on growthinhibition (100-90% growth percent) and apoptosis (5-10% cell death).Nonetheless, the anti-cancer activity of these leads compound 1 wascomparable to the current standard of care, temozolomide, in an in vivoscreen as shown in FIG. 6A wherein compound 1 reduced the level of tumorproliferation relative to the temozolomide.

In vivo studies also confirmed that compound 1 was non-toxic whenadministered systemically. The graph in FIG. 6B shows the percent bodyweight loss verses time for group (N=3 mice) receiving daily systemic(IP) doses of compound 1 for 5 consecutive days. FVB mice (3 months)received the escalating dose of compound 1 for 5 days. When dosing wascompleted, the mice were monitored for 14 days to determine anytoxicity. In mice the best indicator of toxicity is weight loss. Clearlyeven at 20 mg/kg of body weight, the mice were maintaining their weight,indicating a lack of toxicity. Equally important, this in vivo screenconfirmed the results observed in the above described in vitrocell-based activity screens. These data identify that the halogenatedpyrrolo- and thienopyrimidine compounds of the present invention as anew class of agents for the treatment of melanoma

Example 8

Cell proliferation assays for several of the compounds were performed onL1210, a mouse lymphocytic leukemia cell line [38]; CCRF-CEM [39] anacute lymphoblastic leukemia cell line; and HeLa, a human cancer cellline derived from a human cervical adenocarcinoma. [40] Murine LeukemiaL1210, human lymphocytic CEM and human cervix carcinoma HeLa cells wereobtained from the American Type Culture Collection (ATCC) (Rockville,Md.). All assays were performed in 96-well microtiter plates. To eachwell were added (5-7.5)×10⁴ tumor cells and a given amount of the testcompound. The cells were allowed to proliferate for 48 h (murineleukemia L1210 cells) or 72 h (human lymphocytic CEM and human cervixcarcinoma HeLa cells) at 37° C. in a humidified CO₂-controlledatmosphere. At the end of the incubation period, the cells were countedin a Coulter counter. The IC₅₀ (50% inhibitory concentration) wasdefined as the concentration of the compound that inhibited cellproliferation by 50%. The results of this testing, listed in Table 1,shown below, indicate that the dichloro compounds 19 and 20 had apronounced effect on proliferation of the all three cell lines. Presenceof C7 iodine on the 2,4-dichloro pyrrolo[3,2-d] (compound 20) increasesthe anticancer activity by a factor of 5 when compared to compound 19.Replacement of the 2,4-chloro with O-benzyl leads to loss of activity bya factor of 3-15, however the activity of compound 16 is comparable tocompound 19 alluding to compensation of loss of activity due tosubstitution with O-benzyl by iodine at C7. This alludes to a)importance of C7-I towards activity against cancer cell lines for the2,4-O-benzylated analogues (15 and 16).

TABLE 1 L1210 IC₅₀ 6.8 ± 2.8 μM CEM IC₅₀ 25 ± 2.0 μM HeLa IC₅₀ 19 ± 3.0μM

L1210 IC₅₀ 0.93 ± 0.0 μM CEM IC₅₀ 4.9 ± 0.4 μM HeLa IC₅₀ 0.92 ± 0.04 μML1210 IC₅₀ 118 ± 8 μM CEM IC₅₀ 86 ± 22 μM HeLa IC₅₀ 98 ± 14 μM

L1210 IC₅₀ 21 ± 2 μM CEM IC₅₀ 17 ± 4 μM HeLa IC₅₀ 17 ± 0 μM

The same testing regime was conducted on Compounds 1 and 2 to determineif such compounds inhibited tumor cell proliferation with the followingresults:

Example 9

Evaluation of antimicrobial activity in vitro. Compounds were testedagainst the following panel of bacteria and fungi purchased from theAmerican Type Culture Collection (ATCC). Briefly, bacteria were grown tomid-log phase, diluted with fresh medium to an optical density at 600 nm(OD₆₀₀) of 0.030-0.060 and then diluted again 1:10. This suspension (195μL) was added to wells in a 96 well microtiter plate (Sarstedt) and 5 μLof compound dissolved in DMSO was added to give a final concentration of100-0.1 μM at 2.5% DMSO by volume. A DMSO negative control and standardantibiotic positive controls were included in each plate. All compoundswere tested in triplicate for each concentration. Plates were sealedwith parafilm, placed in a Ziploc bag to prevent evaporation, andincubated at 30° C. (fungi) or 37° C. (bacteria) for 16-20 hours (48hours for C. neoformans). The OD₆₀₀ values for each well were determinedwith a plate reader (Biotek, EL800) and the data were standardized tothe DMSO control wells after subtracting the background from blankmedium. Initial single concentrations were tested at 100 μM and activecompounds were further tested with at least nine concentrations for afull dose response. Dose response curves were generated using GraphPadPrism 5 software and used to determine the MIC₉₅ concentrations (minimalconcentration that inhibits 95% of growth). Compounds 1, 2, and 19 werescreened for growth inhibition activity against a panel of bacteria andfungi at a concentration of 100 μM, as shown in Table 2. Compounds 1 and2 exhibited activity against several pathogenic yeast strains (55-99%growth inhibition) and compound 1 also showed weak activity againstBacillus subtilis. In addition, compound 1 displayed a higher potencythan 2 against several clinical strains of Cryptococcus neoformans.

TABLE 2 Antimicrobial screening of thieno[3,2-d]pyrimidines. %inhibition at 100 uM Microbial strain strain designation

Bacterial strains Escherichia coli ATCC 25922 NA NA NA Bacillus subtilisATCC 6633 43% NA NA Staphylococcus ATCC 43300 NA NA NA aureus subsp.aureus (MRSA) Enterococcus ATCC 51299 NA NA NT faecalis (VRE)Pseudomonas ATCC 27853 NA NA NT aeruginosa Fungal strains Candidaalbicans ATCC 10231 99% 99% NA Cryptococcus ATCC 66031 101%  55% NAneoformans C. neoformans JEC20 100%  91% NA C. neoformans VANC-R265 93%67% NA C. neoformans B4546 96% 82% NA NA—No activity, NT: Not tested

Compounds 1 and 2 were further tested against the susceptible fungalstrains to determine the MIC₉₅ values. Although the spectrum ofanti-microbial activity for compound 1 is broad (Table 3), the bromoanalogue 2 is much more selective towards fungi and 2-5 times morepotent than compound 1 (Table 3).

TABLE 3 Antifungal activity of thieno[3,2-d]pyrimidines 1 and 2. MIC₉₅(μM) Strain

C. albicans 23.4 10.7 ATCC 10231 C. neoformans 16.6 5.8 JEC20 C.neoformans 34.6 7.0 B4546 MIC₉₅—minimum concentration for inhibition by95%

Example 10

Cell-cycle and apoptosis studies were conducted in MDA-MB-231 cells.Cell-cycle analysis of compound 19 at 15 μM concentration lead to arrestof 60% cells in G2/M stage. Similarly, at 1.75 μM concentration,compound 20 arrested 30% of cells in G2/M stage. These results allude tothe reduction in ability to arrest cells in G2/M stage upon introductionof iodine at C7. To evaluate the possible mechanism for arrest of cellproliferation, cell-cycle distribution was measured using propidiumiodide staining and flow cytometry. While compound 19 is clearly toxicto MDA-MB-231 cells at the tested concentration, only a modest (althoughstatistically significant) increase in annexin V-positive cells wasdetected. There are several possibilities that could account for this,including: (i) that cell lysis occurs rapidly following induction ofapoptosis, preventing significant accumulation of annexin V-positivebodies, or (ii) that a distinct cell death pathway is triggered bycompound 19. On the other hand treatment with compound 20 induced robustaccumulation of annexin V-positive cells consistent with apoptotic celldeath in a manner that is similar to the effects that were observed forthieno[3,2-d]pyrimidines compound 1 and 2.

This study, besides establishing the antiproliferative properties of the4-Cl pyrrolo[3,2-d]pyrimidines also establishes the effect of C7 iodineon the enhancement of antiproliferative properties; particularly thecytotoxicity manifested by compound 20 against select cancer cell lines.These results have identified antitumor activities of 2,4-dichloropyrrolo[3,2-d]pyrimidines with emphasis on C7 iodine towards enhancingthe potency. These studies establish the use of halogenatedpyrrolo[3,2-d]pyrimidines as pharmacologically useful scaffold. Thesehalogenated compounds manifest antitumor activity against cancer byinducing apoptosis via arrest of cell-cycle in G2/M, as shown in FIG. 9.

Example 11

Many antineoplastic compounds arrest cell proliferation by activatingspecific checkpoints that block progression through the cell cycle. Totest whether compounds 1 or 2 inhibited cell proliferation by thismechanism, their effects on L1210 cell cycle distributions weremonitored using propidium iodide staining and flow cytometry (FIG. 7).Cell cycle and apoptosis studies were conducted on L1210, a mouselymphocytic leukemia cell line obtained from the American Type CultureCollection and grown in Dulbecco's Modified Eagle's Medium (DMEM) plus10% fetal bovine serum (FBS). 2×10⁶ cells were seeded in 100 mm dishesand treated with vehicle alone or compounds 1 or 2 at the IC₅₀ valuesdetermined by cell viability assays or an equivalent volume of DMSO. At24 and 48 h following treatment the cells were collected, fixed, andstained with propidium iodide (Sigma Aldrich) immediately beforeanalysis by flow cytometry. Vehicle-treated cells were largely confinedto G1 and S phases, with very small (<5%) subpopulations in the G2/Mphase or showing sub- (<G1) or super-genomic (>G2) DNA content.Surprisingly, 24 h or 48 h treatment with IC₅₀ concentrations of eithercompound 1 or 2 yielded no substantial changes in L1210 cell cycledistribution, indicating that these reagents do not suppress cell growthby activating cell cycle checkpoint mechanisms. This cell cycle assayand the apoptosis assay discussed in Example 6 indicate that bothcompounds 1 and 2 can induce cell death by an apoptotic-like pathway,but in a manner that does not require arrest at a specific stage of thecell cycle.

Example 12

The thieno[3,2-d]pyrimidine scaffold has been used extensively in thedesign of kinase inhibitors. To determine if the halogenated compoundsof the present invention exhibited the same results, compounds 1 and 2were screened against twenty kinases at 5 μM using Invitrogen's SelectScreen kinase profiling [41] service to explore the possibility ofkinase inhibition. Surprisingly they did not exhibit any substantialinhibitory activity against any of the twenty tested kinases and theresults of such testing are shown in Table 4.

TABLE 4 Inhibition of protein kinases by 2,4-dichlorothieno[3,2-d]pyrimidine at 5 μM % Kinase tested inhibition FRAP1 (mTOR)<40% JAK1, JAK2, JAK2 JHI JH2, JAK2 JHI JH2 V617F, JAK3 <40% LTK (TYK1),TYK2 <40% MET (cMet) <40% PRKCN (PKD3), PRKD1 (PKC mu), PRKD2 (PKD2)<40% SRC, SRC N1 <40% PIK3C2A, PIK3C2B, PIK3C3, PIK3CA/PIK3R1, <40%PIK3CD/PIK3R1, PIK3CG

Example 13

Human metastatic breast cancer MDA-MB-231 cells were obtained from theAmerican Type Culture Collection (ATCC) (Rockville, Md.) and maintainedin Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovineserum (FBS) without antibiotics at 37° C. in a humidified,CO₂-controlled incubator. The cytotoxicity of compounds 1 and 2 wasassessed by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).MDA-MB-231 cells were seeded in 96-well plates at 5×10³ cells per welland treated with a range of drug concentrations. After 48 hours, cellviability was measured using the MTT Cell Proliferation Assay kit (ATCC)according to the manufacturer's instructions. Percent cell viability wasplotted as a function of drug concentrations and analyzed using asigmoidal dose response function with PRISM v3.03 software (GraphPad) toresolve drug concentrations yielding 50% cell death (IC₅₀). The cellcycle distributions of MDA-MB-231 cells treated with compounds 1 or 2were analyzed using propidium iodide staining and flow cytometry. 2×10⁶cells were seeded in 100 mm dishes and treated with compounds 1 or 2 atthe IC₈₀ values determined by cell viability assays or an equivalentvolume of DMSO. 48 h following treatment the cells were collected,fixed, and stained with propidium iodide (Sigma Aldrich) immediatelybefore analysis by flow cytometry.

As shown in Example 11, compounds 1 and 2 both induced apoptosis butinvolving a mechanism independent of cell cycle arrest [42]. To testwhether the toxicity of these agents for MDA-MB-231 cells proceeded viaa similar pathway, their effects were monitored on cell cycledistributions using propidium iodide staining and flow cytometry.Cumulatively over 80% of vehicle-treated MDA-MB-231 cells were confinedto G1 and S phases, with much smaller subpopulations recovered in G2/M(10%) or in cells showing <G1 (<3%) or >G2 (<3%) DNA content (FIG. 8).Treating MDA-MB-231 cells with compound 2 had no effect on their cellcycle distribution, consistent with observations in the L1210 line.However, after treating MDA-MB-231 cells with compound 1, the vastmajority (>70%) accumulated in the G2/M phase of the cell cycle. Cellsubpopulations in G1 and S phases were concomitantly depleted while nochanges were observed in the fraction of cells with <G1 or >G2 DNAcontent. This dramatic accumulation of cells in G2/M indicates thatcompound 1, but not compound 2, triggers mitotic arrest in MDA-MB-231cells.

Example 14

The lead halogenated pyrrolopyrimidines and thienopyrimidines of thepresent invention were tested in the human cell line screen which iscomposed of 60 different human tumor cell lines (NCI-60) representingleukemia, melanoma, lung cancer, colon cancer, brain cancer, ovarycancer, breast cancer, prostate cancer and kidney cancer. Testingspecifics can be found at the National Cancer Institute athttp://dtp.nci.nih.gov/branches/btb/ivclsp.html. Compounds 1, 2 and 19were tested initially at a single dose (10⁻⁵M) in the full cell panel.The results of the one-dose are reported as a mean graph of the percentof the growth of the treated cells. Both growth inhibition (valuesbetween 0 and 100) and lethality (values less than 0) are detectable. Toevaluate the test results, a value of 100 means no growth inhibition. Avalue of 40 would mean 60% growth inhibition. A value of zero means nonet growth over the course of the experiment. A value of −40 means 40%lethality and a value of −100 means all the cells are dead. FIG. 10shows the one dose results for compound 1, FIG. 11 shows the one doseresults of compound 2 and FIG. 12 shows the one dose results of compound19. The cells with high lethality are emphasized with an arrow in theone dose compilation of results shown in FIGS. 10, 11 and 12. Theresults of the 5 dose screening are shown in FIG. 13.

Example 15

In order to investigate the PK profile of these compounds, a series ofhalogenated pyrrolo[3,2-d]pyrimidine compounds was synthesized and firsttested for activity in various cancer cell lines followed by a mousemodel. EC50 values ranged from 0.014-14.5 μM, and maximum tolerateddoses (MTD) in mice were between 5-10 mg/kg. This indicates a widevariance in activity and toxicity that necessitated the synthesis of asecond series of compounds with N5 alkyl substitutions in an effort toslow the rate of metabolism, which was thought to be leading to thetoxicity.

Specifically, in an effort to further pursue efficaciouspyrrolopyrimidines with antiproliferative properties, the synthesis of aseries of compounds with modifications to the pyrrole ring in thepyrrolo[3,2-d]pyrimidine scaffold was undertaken. The effect of variouspyrrole substituents on compound pharmacokinetics were examined, as wellas the compounds' efficacy against a wide range of cancer cell lines.The target compounds are shown in below:

2,4-Dichloro-7-iodo-5-tosyl-pyrrolo[3,2-d]pyrimidine (9)

2,4-dichloro-7-iodo-5H-pyrrolo[3,2-d]pyrimidine (100 mg, 0.318 mmol) wasdissolved in 1:1 THF:DMF (anhydrous) at room temperature under N₂. NaH(60% in mineral oil, 16 mg, 0.398 mmol) was added directly and reactionstirred for 1.5 h. p-Toluenesulfonyl chloride (76 mg, 0.398 mmol) wasadded and the reaction stirred overnight. The solvent was removed, andthe residue dissolved in EtOAc and washed with water and brine, thendried over MgSO₄. Organics were loaded onto Celite and product purifiedusing silica column chromatography eluting with 5:1 hexanes:EtOAc toobtain a white powder (126 mg, 85%). ¹H NMR (400 MHz, CDCl₃): δ8.82 (s,1H), 67.94-7.92 (d, 2H, J=8 Hz), 67.46-7.44 (d, 2H, J=8 Hz), δ2.43 (s,3H). ¹³C NMR (100 MHZ, CDCl₃): δ158.1, 154.2, 146.9, 140.9, 137.1,134.5, 130.5, 127.9, 64.1, 21.9. APCI-MS m/z for C₁₃H₈Cl₂IN₃O₂S calcd.at 466.9, found 467.97 (M+H). Anal. calcd. for C₁₄H₈Cl₂IN₃O₂S: C, 33.36;H, 1.72; N, 8.98. Found: C, 33.36; H, 1.74; N, 8.96.

5-((Benzyloxy)methyl)-2,4-dichloro-7-iodo-pyrrolo[3,2-d]pyrimidine (8)

2, 4-dichloro-7-iodo-5H-pyrrolo[3,2-d]pyrimidine (100 mg, 0.318 mmol)was dissolved in 1:1 THF:DMF (anhydrous) at room temperature under N₂.NaH (60% in mineral oil, 16 mg, 0.398 mmol) was added directly andreaction stirred for 1.5 h. Benzyl chloromethyl ether (73 μL, 0.398mmol) was added and reaction stirred for 3 h, upon which TLC indicatedreaction completion. The solvent was removed and residue dissolved inEtOAc and washed with water and brine, then dried over MgSO₄. Organicswere loaded onto Celite and product purified using silica columnchromatography eluting with 5:1 hexanes:EtOAc to obtain a white powder(124 mg, 90%). ¹H NMR (400 MHz, CDCl₃): δ 7.63 (s, 1H), δ7.29-7.28 (m,3H), δ7.25-7.22 (m, 2H), δ5.79 (s, 2H), δ4.54 (s, 2H). ¹³C NMR (100 MHZ,CDCl₃): δ 155.4, 150.4, 146.1, 144.8, 143.6, 128.5, 128.1, 127.9, 123.6,86.1, 77.9, 70.4, 58.8. APCI-MS m/z for C₁₄H₁₀Cl₂IN₃O calcd. at 432.92,found 434.06 (M+H). Anal. calcd. for C₁₄H₁₀Cl₂IN₃O: C, 38.74; H, 2.32;N, 9.68. Found: C, 38.74; H, 2.34; N, 9.67.

The highest activity was against MIA Pa-Ca-2 pancreatic cancer cells bycompound 6 as shown below in Table 5. However, compound 7 also displayedsubmicromolar activity and was subsequently determined to have decreasedtoxicity compared at 6. These combined traits led us to utilize 7 as alead compound, with prodrug formulation through substitution at N5. Theactivity of prodrug 8 decreased ten-fold compared to the parent, but itwas observed that 9 was comparable (Table 5). Previous work on thepyrrolopyrimidine analogues had indicated that the presence of a halogenat C4 on the pyrimidine was essential for activity and that the presenceof a halogen at C7 enhanced the activity—a trend also observed in thecurrent study.

TABLE 5 Cancer cell line activity screening for target compounds 5-7.Com- MDA- MIA Pa—Ca pound A549 MB-231 2 MOLM14 5 14.5 ± 3.5  9.7 ± 9.75.3 ± 1.0 9.5 ± 3.1 6 0.05 ± 0.02 0.4 ± 0.3 0.014 ± 0.001 0.023 ± 0.0047 1.0 ± 0.1 1.2 ± 0.4 0.37 ± 0.05 0.55 ± 0.07 8 ND^(b) 6.1 ± 0.4 7.3 ±0.8 ND 9 ND  0.79 ± 0.04  0.5 ± 0.02 ND Values shown in μM, ND = notdetermined

The maximum tolerated dose (MTD) for compounds 6 and 7 was 5 and 10mg/kg, respectively as shown below in Table 6. The higher toxicity ofcompound 6 excluded it as a lead compound. In addition, although thetoxicity of 6 is only 2-fold higher than 7, significant mortality ratewas observed in the mouse model immediately following administration,raising concerns that mortality would interfere with effective dosestudies. The MTD of compound 7 is within a comparative range of similarcompounds and still exhibited low micromolar activity, and as such, itwas chosen for further modifications in an attempt to decrease toxicity.The addition of a toluenesulfonyl (tos) group in 9 raised the MTD from10 to 40 mg/kg, while maintaining submicromolar levels of activityagainst all tested cell lines.

TABLE 6 Maximum tolerated dose and plasma half-life of compounds 6, 7,and 9. Compound MTD^(a) t_(1/2) 6 5 ND^(b) 7 10 30.7 min 9 40 32.7 minMTD values shown in mg/kg. ND = not determined

Further, mouse studies on 7 were carried out at The Cancer CenterPharmacology Core (CCPC, Colorado State University) by injection of 10mg/kg of the compound, followed by euthanization at 15, 30, 60, 90, 120,240, and 480 minutes. It was found that the compound was rapidlymetabolized in vivo, with a half-life of 30.7 minutes (Table 6). Mousestudies on 9 were performed in the same manner. The half-life of 9 issimilar to the parent compound, at 32.7 minutes (Table 6).

Plasma stability was carried out at the CCPC by spiking mouse plasmawith 500 ng/mL of 9 and analyzing for both 9 and 7 at 0, 5, 15, 30, 45,60, and 90 minutes. Significant conversion of 9 to 7 over the time frameof the experiment was observed as shown in

FIG. 14. It is theorized and the results show a strong indication thatplasma enzymes are likely capable of metabolizing the prodrug to itsactive form. Supporting this hypothesis is the analysis of 7 in plasma,which shows excellent stability with a deviation of ±5.7% over the timeframe investigated.

Initial screening of 5-7 against six cancer cell lines showed IC₅₀values as low as 0.014±0.0 μM (Table 5). All of the compoundsdemonstrated activity against all four cell lines, indicating that theyhave potential as broad spectrum antiproliferative agents. However, thebest activity overall was displayed against MIA Pa-Ca-2 pancreaticcancer cells.

Maximum tolerated dose of 6, 7, and 9 was between 5-40 mg/kg, indicativeof high toxicity but not outside the range of current epigenetictherapeutics such as paclitaxel (25 mg/kg), docetaxel (20 mg/kg) andvinorelbine (2.5 mg/kg), as well as experimental antiproliferativecompound LY231514 (5 mg/kg). However, as these other clinical compoundsare all semi-synthetic, complex molecules with different mechanisms ofaction than DNA damage, it is difficult to compare clinical resultsbetween them and the fully synthetic pyrrolo[3,2-d]pyrimidines of thepresent invention.

The parent compound 7 displays rapid pharmacokinetics, with little to nodetection of the compound in mouse plasma within 70 minutes. Prodrugs 8and 9 were designed to decrease the metabolic rate by both stericallyhindering access to the reactive C4 and C7 positions as well as byacting as a labile leaving group following metabolism to the activecompound 7. Prodrug 9 proved stable in aqueous buffer for up to 24hours, however, was undetectable in biological media within 1 hour. Apharmacokinetic profile supported the serum stability studies,indicating a half-life 30.7 minutes for compound 7 and a half-life of32.7 minutes for 9. The plasma studies confirm that the breakdownproduct of 9 is the active compound 7 as shown in FIG. 14.

The N-substituted compounds demonstrated comparable cell line activity(EC50 values between 0.83-7.3 μM) with significantly decreased toxicity(MTD=40 mg/kg). Finally, the PK profile of the active N5-substitutedcompound shows a plasma half-life of 32.7 minutes, and rapid conversioninto the parent unsubstituted analogue. Together, these data indicatethat halogenated pyrrolo[3,2-d]pyrimidines present a promising lead intopotent antiproliferative agents with tunable activity and toxicity, andrapid metabolism.

Halogenated pyrrolo[2,3-d]pyrimidine analogues have been shown to bepotent antiproliferative agents against a wide spectrum of cancer celllines. The effective dose of these compounds is in the low micromolar tonanomolar range, representing a promising avenue of lead compounds. Twodisadvantages for these compounds are their toxicity and rapidmetabolism. However, by manipulation of the prodrug moieties at the N5position, as shown herein, both of these have been remediatedsignificantly. Clearly, the modifications shown herein can potentiallydecrease the toxicity and improve the pharmacokinetic profile. Thisallows the prodrugs to be more effectively delivered to the target cellsin vivo before undergoing metabolism to their active form.

General Procedure for Synthesis of Compounds 23-32

2,4-dichloro-1H-pyrrolo[3,2-d]pyridine was dissolved in a solution of1:1 THF:DMF under N₂. Sodium hydride (60% suspension in oil) was addeddirectly, and the mixture was stirred at room temperature for 1 h.Appropriate halogenated reagent (methanesulfonyl chloride (3),2-nitrobenzenesulfonyl chloride (4), 2,4,6-Trii sopropylbenzenesulfonylchloride (5), benzyl bromide (6), 2,4-dichlorobenzyl bromide (7),4-methoxybenzyl chloride (8), acetyl chloride (9), isobutyryl chloride(10), diphenylcarbamyl chloride (11), iodoethane (12)) was added and themixture was stirred at r.t. for 18 h. The solvent was removed andresidue dissolved in EtOAc and washed with water and brine, then driedover MgSO₄. Organics were loaded onto Celite and product purified usingsilica column chromatography eluting with 4:1 hexanes:EtOAc or 9:1DCM:MeOH to obtain product, generally as a white to off-white solid.

2,4-dichloro-5-(methylsulfonyl)-5H-pyrrolo[3,2-d]pyrimidine (23)

¹H-NMR (CDCl₃) (ppm) δ: 3.66 (s, 3H); 6.83 (d, J=4.12, 1H); 8.10 (d,J=4.12, 1H). ¹³C NMR (100 MHZ, DMSO-d₆): δ 158.79, 153.40, 152.14,144.44, 139.10, 137.58, 124.23, 123.05, 105.97, 102.31, 44.88. ESI-MSm/z for C₇H₅C1₂N₃O₂S calcd. at 264.95, found at 266.01 (M+H)⁺. Anal.calcd. for C₇H₅Cl₂N₃O₂S: C, 31.60; H, 1.89; N, 15.79. Found: C, 31.58;H, 1.89; N, 15.78.

2,4-dichloro-5-((2-nitrophenyl)sulfonyl)-5H-pyrrolo[3,2-d] pyrimidine(24)

¹H-NMR (DMSO-d₆) (ppm) δ: 7.20 (t, J=2.32, 1H); 7.75 (d, J=8.24, 1H);7.82 (t, J=7.80, 1H); 8.04 (q, J=7.32, 1H); 8.25 (d, J=7.80, 1H); 8.60(d, J=2.76, 1H). ¹³C NMR (100 MHZ, DMSO-d₆): δ 158.30, 152.97, 147.51,144.41, 140.13, 137.28, 134.88, 131.37, 130.61, 126.68, 108.04, 102.79.ESI-MS m/z for C₁₂H₆Cl₂N₄O₄S calcd. at 371.95, found at 373.00 (M+H)⁺.Anal. calcd. for C₁₂H₆Cl₂N₄O₄S: C, 38.62; H, 1.62; N, 15.01. Found: C,38.61; H, 1.62; N, 15.01.

2,4-dichloro-5-((2,4,6-triisopropylphenyl)sulfonyl)-5H-pyrrolo[3,2-d]pyrimidine(25)

¹H-NMR (CDCl₃) (ppm) δ: 1.08 (d, J=6.84, 12H); 1.24 (d, J=6.88, 6H);2.92 (septet, 1H); 3.85 (septet, 2H); 6.83 (d, J=3.64, 1H); 7.17 (s,2H); 8.26 (d, J=3.68, 1H). ¹³C NMR (100 MHZ, CDCl₃): δ157.78, 155.99,153.18, 151.22, 145.17, 136.98, 131.89, 124.12, 123.62, 105.89, 34.42,30.26, 24.38, 23.55. ESI-MS m/z for C₂₁H₂₅Cl₂N₃O₂S calcd. at 453.10,found at 454.10 (M+H)⁺. Anal. calcd. for C₂₁H₂₅Cl₂N₃O₂S: C, 55.51; H,5.55; N, 9.25. Found: C, 55.49; H, 5.54; N, 9.24.

2,4-dichloro-5-benzyl-5H-pyrrolo[3,2-d]pyrimidine (26)

¹H-NMR (CDCl₃) (ppm) δ: 5.67 (s, 2H); 6.71 (d, J=3.2, 1H); 7.05 (d,J=6.44, 2H); 7.34 (m, 3H); 7.53 (d, J=3.2, 1H). ¹³C NMR (100 MHZ,CDCl₃): 154.4,4, 150.61, 143.16, 138.92, 136.36, 129.25, 128.48, 126.63,123.15, 103.00, 52.41. ESI-MS m/z for C₁₃H₉Cl₂N₃ calcd. at 277.02, foundat 278.03 (M+H)⁺. Anal. calcd. for C₁₃H₉Cl₂N₃: C, 56.14; H, 3.26; N,15.11. Found: C, 56.11; H, 3.26; N, 15.10.

2,4-dichloro-5-(2,4-dichlorobenzyl)-5H-pyrrolo[3,2-d]pyrimidine (27)

¹H-NMR (CDCl₃) (ppm) δ: 5.72 (s, 2H); 6.42 (d, J=8.68, 1H); 6.75 (d,J=3.24, 1H); 7.15 (d, J=8.28, 1H); 7.46 (d, J=2.28, 1H); 7.51 (d J=3.20,1H). ¹³C NMR (100 MHZ, DMSO-d₆): δ154.39, 149.55, 142.77, 142.23,135.14, 133.24, 132.01, 129.52, 128.13, 122.50, 103.03, 49.67. ESI-MSm/z for C₁₃H₇Cl₄N₃ calcd. at 344.94, found at 345.95 (M+H)⁺. Anal.calcd. for C₁₃H₇Cl₄N₃: C, 45.00; H, 2.03; N, 12.11. Found: C, 44.96; H,2.03; N, 12.09.

2,4-dichloro-5-(4-methoxybenzyl)-5H-pyrrolo[3,2-d]pyrimidine (28)

¹H-NMR (CDCl₃) (ppm) δ: 3.78 (t, J=7.36, 3H); 5.60 (d, J=3.68, 2H); 6.67(s, 1H); 6.86 (q, J=4.12, 2H); 7.03 (t, J=3.68, 2H); 7.50 (s, 1H). ¹³CNMR (100 MHZ, DMSO-d₆): δ159.48, 155.10, 149.01, 142.96, 141.49, 141.00,130.06, 128.35, 123.01, 114.97, 102.07, 55.61, 51.20. ESI-MS m/z forC₁₄H₁₁Cl₂N₃O calcd. at 307.03, found at 307.93 (M+H)⁺. Anal. calcd. forC₁₄H₁₁Cl₂N₃O: C, 54.57; H, 3.60; N, 13.64. Found: C, 54.54; H, 3.60; N,13.63.

1-(2,4-dichloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)ethan-1-one (29)

¹H-NMR (CDCl₃) (ppm) δ: 2.73 (s, 3H); 6.77 (d, J=3.64, 1H); 7.92 (d,J=4.12, 1H). ¹³C NMR (100 MHZ, DMSO-d₆): δ 168.26, 158.29, 152.17,145.87, 140.28, 123.01, 107.93, 24.18. ESI-MS m/z for C₈H₅Cl₂N₃O calcd.at 228.98, found at 229.54 (M+H)⁺. Anal. calcd. for C₈H₅Cl₂N₃O: C,41.77; H, 12.19; N, 18.27. Found: C, 41.75; H, 2.19; N, 18.25.

1-(2,4-dichloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)-2-methylpropan-1-one(30)

¹H-NMR (CDCl₃) (ppm) δ: 1.37 (d, J=6.84, 6H); 3.33 (septet, J=6.88, 1H);6.77 (d, J=3.68, 1H); 7.92 (d, J=3.64, 1H). ¹³C NMR (100 MHZ, DMSO-d₆):δ174.60, 158.78, 152.44, 146.37, 138.82, 124.21, 106.45, 34.17, 19.07.ESI-MS m/z for C₁₀H₉Cl₂N═O calcd. at 257.01, found at 257.68 (M+H)⁺.Anal. calcd. for C₁₀H₉Cl₂N₃O: C, 46.54; H, 3.51; N, 16.28. Found: C,46.51; H, 3.51; N, 16.27.

2,4-dichloro-N,N-diphenyl-5H-pyrrolo[3,2-d]pyrimidine-5-carboxamide (31)

¹H-NMR (DMSO-d₆) (ppm) δ: 6.76 (d, J=3.20, 1H); 7.23-7.34 (m, 10H); 8.37(d, J=3.20, 1H). ¹³C NMR (100 MHZ, DMSO-d₆): δ 155.30, 151.12, 149.65,144.15, 141.73, 139.55, 130.09, 123.10, 104.63. ESI-MS m/z forC₁₉H₁₂Cl₂N₄O calcd. at 382.04, found at 383.04 (M+H)⁺. Anal. calcd. forC₁₉H₁₂Cl₂N₄O: C, 59.55; H, 3.16; N, 14.62. Found: C, 59.53; H, 3.16; N,14.61.

2-(2,4-dichloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)ethan-1-ol (32)

¹H-NMR (DMSO-d₆) (ppm) δ: 3.70 (d, J=5.52, 2H); 4.50 (t, J=5.48, 2H);6.68 (d, J=3.24, 1H); 8.05 (d, J=3.20, 1H); 8.08 (s, 1H). ¹³C NMR (100MHZ, DMSO-d₆): δ 153.40, 142.99, 137.59, 124.25, 102.56, 101.54, 61.40,51.47. ESI-MS m/z for C₈H₇Cl₂N₃O calcd. at 231.00, found at 231.93(M+H)⁺.

REFERENCES

The contents of all references cited herein are incorporated byreference herein for all purposes.

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That which is claimed is:
 1. A method of inducing apoptosis and/orinhibiting the growth of cancer cells, the method comprisingadministering to a subject in need of such treatment a therapeuticallyeffective amount of a prodrug of a halogenated pyrrolopyrimidine orthienopyrimidine compound having the following formula:

wherein X is S or NH and Y is H, Br or I.
 2. The method of claim 1,wherein the cancer cells are select from breast, colon, renal, melanomaand leukemia cancer cells:
 3. The method of claim 1, wherein the prodrugis administered to the subject.
 4. The method of claim 1, wherein theprodrug is converted, by a chemical or physiological process in theindividual to the halogenated pyrrolopyrimidine or thienopyrimidinecompound.
 5. A method of inducing apoptosis and/or inhibiting the growthof cancer cells, the method comprising administering to a subject inneed of such treatment a therapeutically effective amount of a prodrugof a halogenated pyrrolopyrimidine compound or halogenatedthienopyrimidine compound having the following formula:

wherein R¹ and R² are selected from R¹=BOM, R²═I; R¹=Tos, R²═I; and R¹is selected from the group of protective groups consisting of:

R² is H, Br or I, wherein BOM is benzyloxymethyl and Tos isp-toluenesulfonyl group.
 6. The method of claim 5, wherein R¹ istert-Butoxy carbamate (Boc), 9-Fluorenylmethoxycarbonyl (Fmoc),Methoxymethyl acetal (Mom), Benzyl, or Acetyl protection.
 7. The methodof claim 5, wherein the cancer cells are select from breast, colon,renal, melanoma and leukemia cancer cells:
 8. The method of claim 5,wherein the prodrug has the following structure:


9. The method of claim 5, wherein the prodrug is converted, by achemical or physiological process in the individual to the halogenatedpyrrolopyrimidine or thienopyrimidine compound.
 10. A method ofinhibiting cancer proliferation activity and the use thereof for cancertreatment in an individual, the methods involving administering to theindividual a prodrug having the following structure;

that is converted in the individual to the following compound


11. The method of claim 10, wherein the prodrug is converted, by achemical or physiological process in the individual to the halogenatedpyrrolopyrimidine compound.
 12. The method of claim 10, wherein thecancer cells are select from breast, colon, renal, melanoma and leukemiacancer cells.